NR AJOK
AU Post,K.; Riesner,D.; Walldorf,V.; Mehlhorn,H.
TI Fly larvae and pupae as vectors for scrapie
QU Lancet 1999 Dec 4; 354(9194): 1969-70
IA http://www.thelancet.com/newlancet/sub/issues/vol354no9194/body.research1969.html
PT letter
AB We analysed experimental transmissibility of the scrapie agent by natural vectors. A fly, Sacrophaga carnaria, fed with brains of scrapie-infected hamsters in different developmental stages caused scrapie in hamsters after they ate fly extracts.
VT
According to the prion model the scrapie agent consists mainly if not entirely of an abnormal isoform (PrPsc) of the cellular prion protein (PrPc).[1] The mode of natural transmission of the infectious agent is unknown, although a scrapie-free flock of sheep developed scrapie after being in contact with a scrapie-positive flock,[2] which led to the hypothesis that there may be a reservoir for scrapie infectivity in fields. Attempts to transmit scrapie by nematodes has failed,[3] but analysis of hay mites from scrapie-affected farms revealed infected mites.[4] To analyse natural vector systems, larvae of meat-eating and myiasis-causing flies (Sarcophaga carnaria) and grass mites were exposed to scrapie infectious material. They were tested for their potential to accumulate PrPsc as determined by Western blotting and their ability to transmit scrapie in the hamster model.
16 larvae of S carnaria were fed with 2 mg of either brains of scrapie-infected hamsters (scrapie strain 263K) or healthy control brains. Every 2 days, larvae and pupae, respectively, were assayed for the presence of Proteinase K (PK) resistant PrP. Samples of S carnaria were washed, separated from their outer cuticula and homogenised in TBS-buffer. The homogenates were analysed for PrP with and without PK-digestion and western blotting.[5] 2 days after eating PrPsc positive brains, larvae showed high amounts of PK-resistant PrP whereas in larvae eating healthy control brains no PrP was detectable. Several days later, neither in living larvae having eaten PrPsc nor in the control group was PrP detectable. However, in infected larvae who died, PK-resistant PrP was detectable after 14 days (data not shown), indicating a high stability of PrP and supposed conservation of infectivity after endogenous metabolism ended. In separate studies, the inner organs of six third instar larvae, fed with PrPsc infected brains for 2 days, were dissolved in 3 ml 0.85% NaCI and given by oesophageal tube to eight hamsters. 10 days after being fed infected brains, six fly pupae were given orally to four hamsters. In another study, 200 oribatid and other mites were exposed to infected hamster brain in a glass bottle for 10 days; 60 mites were dissolved in 1 mL 0.85% NaCI and given orally to two hamsters. As a control for all transmission studies infected hamster brain was homogenised in 1 mL 0.85% NaCI and given orally to four hamsters. Animals were observed regularly and killed when they showed signs of scrapie for assay for PrPsc by PK-digestion and western blotting. All remaining hamsters were killed 215 days after the start of the experiment and also examined.
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Distribution of infectivity and PrPsc of orally-infected hamsters
Source of Number of Incubation Number of
infectious clinically ill time, days PrPsc-positive
material hamsters/total hamsters/total
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Larvae contents 6/8 165, 189*, 189, 5/8
201, 201, >215+
Pupae contents 2/4 189, >215+ 3§/4
Mites 0/2 - 0/2
Hamster brain 2/4 144, 189 2/4
* animal developed other disease than scrapie, no PrPsc detectable.
+ hamster clinically suspect, showing first signs of scrapie, both PrPsc positive.
§ hamster without symptoms, but PrPsc positive
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The table shows the distribution of infectivity as determined by incubation time, the number of animals with signs of scrapie, and the appearance of PrPsc. Six of eight hamsters, orally inoculated with larvae contents, became ill, but only five of them were PrPsc positive. Two of four pupae-contents-inoculated hamsters developed clinical signs of scrapie, and three were positive for PrPsc. None of both hamsters inoculated with mites showed clinical signs of scrapie or PrPsc. Two of four hamsters infected with brain material developed scrapie within short incubation times and were PrPsc positive. The irregular distribution of infectivity results probably from incomplete homogenation of the inoculum.
We conclude that the contents of larvae and pupae from flies having eaten infectious hamster brains can transmit scrapie. This strongly suggest a conservation of infectivity in larvae and pupae. Whether they replicate infectivity as postulated by Wisniewski and colleagues[4] is unknown and appears improbable because no PrP-encoding gene in any insect has been reported. Our transmission experiments using mites failed. Probably the number of mites inoculated was too low. The findings of the present work suggest that prion diseases might be transmitted by flies in different development stages, even after death. Our results may be relevant also to transmission of bovine spongiform encephalopathy.
1 Prusiner SB. Prions. Proc Natl Acad Sci USA 1998; 95: 13363-83.
2 Hoinville LJ. A review of the epidemiology of scrapie in sheep. Rev Sci Tech Off Int Epiz 1996; 15: 827-52.
3 Fitzsimmons WM, Pattison IH. Unsuccessful attempts to transmit scrapie by nematode parasites. Res vet Sci 1968; 9: 281-83.
4 Wisniewski HM, Sigurdarson S, Rubenstein R, Kascsak RJ, Carp RI. Mites as vectors for scrapie. Lancet 1996; 347: 1114.
5 Beekes M, Baldauf E, Caßens S, et al. Western blot mapping of disease-specific amyloid in various animal and human with transmissible spongiform encephalopathies using a high yield purification method. J Gen Virol 1995; 76: 2567-76.
Institut für Physikalische Biologie (K Post PhD, D Riesner PhD), Institut für Parasitologie (V Walldorf PhD) and Biologisch-Medizinisches Forschungszentrum (H Mehlhorn PhD), Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
IN
Auf ungefähr 2 g Hirn gesunder Hamster und auf die gleiche Menge Hirn von mit dem Scrapiestamm 263K infizierten Hamstern wurden je 16 fleischfressende Larven der Fliege Sacrophaga carnaria gesetzt. Nach etwa 1 Tag hatten diese das Hirngewebe gefressen. Alle 2 Tage wurden einige Larven bzw. Puppen gewaschen, aus ihrer äußeren Hülle geholt und in Puffer homogenisiert, um darin Protease-resistentes Prionprotein nachzuweisen.
Zwei Tage nach dem Beginn oder dem Ende der Fütterung mit infektiösem Hirn (der Artikel beschreibt dies nicht eindeutig) enthielten die Larven große Mengen PrPsc, während in den mit nicht infiziertem Gehirn gefüttertem Kontrolltieren kein Protease-resistentes Prionprotein nachweisbar war. Mehrere Tage später war auch in den mit PrPsc gefütterten Larven kein PrPsc mehr nachweisbar, sofern diese nicht schon vor der Probennahme gestorben waren. In gestorbenen Larven war PrPsc noch nach 14 Tagen nachweisbar. Die lebenden Fliegenlarven waren also in der Lage, PrPsc aktiv abzubauen oder auszuscheiden, während dieses in toten Larven stabil war.
In zwei weiteren Experimenten wurden je 6 Fliegenlarven im dritten Larvenstadium 2 Tage lang mit Scrapie-infektiösem Hirn gefüttert. Im einen Fall direkt und im anderen Fall erst nach 10 Tagen wurden ihre inneren Organe in 3 ml 0,85%-iger Kochsalzlösung homogenisiert . Diese Suspensionen wurden im ersten Fall 8 Hamstern, im zweiten Fall 4 Hamstern direkt in den Magen gespritzt. Es erkrankten 6 der 8 und 2 der 4 Hamster. PrPsc war in 5 der 8 und in 3 der 4 Hamster nachweisbar. Die Infektiosität blieb also zumindest 10 Tage lang in den Fliegenlarven bzw. Puppen erhalten.
In einem weiteren Experiment wurde 200 Hornmilben und anderen Milben in einem Glas 10 Tage lang infektiöses Hamsterhirn angeboten. 60 Milben wurden in 1 ml 0,85%-iger Kochsalzlösung homogenisiert, um damit 2 Hamster oral zu inokulieren. Keiner der beiden Hamster erkrankte binnen 215 Tagen und nach diesem Zeitraum wurde auch kein PrPsc gefunden.
Als Positivkontrolle wurde auch eine leider nicht genannte Menge infektiösen Hamsterhirns in 1 ml 0,85%-iger Kochsalzlösung homogenisiert, um damit 4 Hamstern oral zu inokulieren. Es erkrankten nur 2 der 4 Hamster dieser Positivkontrolle und es war auch nur in diesen beiden Hamstern PrPsc nachweisbar.
Alle Empfänger-Hamster wurden regelmäßig beobachtet und nach dem Auftreten von Scrapie-Symptomen oder spätestens nach 215 Tagen getötet, um dann per Western blot nach PrPsc zu suchen.
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Verteilung von Infektiosität und PrPsc in oral infizierten Hamstern
Quelle des Anteil der Inkuba- Anteil der
infektiösen erkrankten tionszeit PrPsc-positiven
Materials Hamster in Tagen Hamster
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Larveninhalte 6/8 165, 189*, 189, 5/8
201, 201, >215+
Puppeninhalte 2/4 189, >215+ 3§/4
Milben 0/2 - 0/2
Hamsterhirn 2/4 144, 189 2/4
* Das Tier bekam eine andere Krankheit und man fand kein PrPsc.
+ Der jeweilige Hamster entwickelte Scrapie-verdächtige Symptome und nachweisbare Mengen von PrPsc.
§ Hamster ohne Symptome, aber PrPsc-positiv
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Die Tabelle zeigt für jedes Inokulum, wieviele Empfänger-Hamster nach welchen Inkubationszeiten erkrankten und bei wievielen PrPsc nachweisbar war.
MH Animal; Diptera/chemistry/growth & development/*physiology; Endopeptidase K; Hamsters; *Insect Vectors; Larva; Mites/physiology; PrPc Proteins/analysis; PrPsc Proteins/analysis; Pupa; Scrapie/*transmission
AD Karin Post, Detlev Riesner, Institut für Physikalische Biologie; Volker Walldorf, Institut für Parasitologie; Heinz Mehlhorn, Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
SP englisch
PO England
EA pdf-Datei und HTML-Version
ZF kritische Zusammenfassung von Roland Heynkes